I. Field of the Invention
The present invention relates to the field of cancer biology. More specifically, the present invention provides methods for the diagnosis of cancer and for the screening of substances that can be used to treat cancer. A particular embodiment of the present invention involves an assay for the detection of telomerase activity in a patient tissue sample.
II. Related Art
Cancers are one of the leading causes of mortality in the world, being responsible for over one-half million deaths in the United States each year. Prompt clinical intervention is important in lowering the mortality rate and, therefore, it is important to provide improved methods for detecting cancers, especially at relative early stages.
One of the new areas of interest in tumor biology relates to the role of telomeres in cellular growth control. Telomeres are repeated sequences found at chromosome ends and it has long been known that chromosomes with truncated ends are unstable, tend to fuse with other chromosomes and are otherwise lost during cell division. Some data suggest that telomeres interaction the nucleoprotein complex and the nuclear matrix. One putative role for telomeres includes stabilizing chromosomes and shielding the ends from degradative enzyme.
Another possible role for telomeres is in replication. According to present doctrine, replication of DNA requires starts from short RNA primers annealed to the 3'-end of the template. The result of this mechanism is an "end replication problem" in which the region corresponding to the RNA primer is not replicated. Over many cell divisions, this will result in the progressive truncation of the chromosome. It is thought that telomeres may provide a buffer against this effect, at least until they are themselves eliminated by this effect.
Thus, the loss of telomeres may be part of programmed cell senescence and, conversely, the maintenance of telomeres may correspond to cell growth. These structures are maintained by an enzyme called telomerase. Telomerase contains RNA and protein components that act to synthesize telomeric sequences. If abnormal maintenance or proliferation of telomeres occurred, it is conceivable that unrestricted, i.e., neoplastic cell growth might result. Recently, there have been numerous reports lending credibility to this theory. For example, 94% of neuroblastomas tested in one study showed telomerase activity while normal adrenal tissue and benign ganglioneuromas did not (Hiyama et al., 1995). Similarly, Hiyama et al. (1995) found that over 80% of primary lung cancer tissues had telomerase activity while only 4.4% of normal tissues had activity. Chadeneau et al., (1995) found telomerase activity in colorectal cancer, but not in edematous polyps and Counter et al. (1994) reported telomerase activity in ovarian carcinoma. Abnormal telomerase activity also has been observed in tumors derived from skin, adipose, connective tissue, breast, stomach, pancreas, cervix, kidney, bladder, colon, prostate and blood (Kim et al., 1994).
Telomerase activity can be assayed by standard methods, which incorporate radioactive nucleotides into a substrate for telomerase followed by gel electrophoresis for analysis of the products formed. This conventional assay is difficult and insensitive, requiring about 10.sup.7 cells per assay (Morin, 1989). There are two known forms of telomerase--processive and non-processive. Processive telomerase adds multiple telomeric repeats to a DNA primer, while non-processive telomerase adds no more than one telomeric repeat to a DNA primer.
Kim et al. (1994) reported improved methods for the extraction and detection of telomerase activity. A detergent lysis method was modified to allow more uniform extraction of telomerase activity, even at low cell numbers. In addition, a PCR-based assay known as "TRAP" (telomeric repeat amplification protocol) was developed. This assay uses telomerase activity in a sample to generate telomerase-specific products which act as templates for amplification by polymerase.
Kim et al. (1994) allude to problems with respect to the PCR products generated. "Even under high stringency, staggered annealing of the downstream primer occurred (for example, annealing by three of the four repeats) . . . Thus, TRAP assay products do not directly reflect the distribution of telomerase products generated in the assay, and the interaction between the upstream and downstream primers must be prevented." As a result, TRAP can produce false-positive results and is not very quantitative. Additionally, the TRAP assay cannot distinguish or even properly detect non-processive telomerase activity. Therefore, there remains a need for a more reliable assay for telomerase activity in tissue samples.